The development of a DNA diagnostic assay for the rapid detection of rifampin-resistant (Rif) Mycobacterium tuberculosis strains directly from patient specimens could result in a decrease in the amount of time from sample acquisition to diagnosis of resistance in tuberculosis patients. This could lead to implementation of appropriate drug therapy much earlier in the course of disease, and, thereby, increase patient's chances of survival, potentially decrease the spread of drug-resistant tuberculosis and decrease the cost of treatment of affected individuals. The identification of the molecular mechanism of rifampin resistance in M. tuberculosis is an important step in the development of a DNA diagnostic assay for the rapid detection of these organisms directly from patient samples and in choosing rational approaches for alternative drug design. The work outlined in this proposed research is designed to determine the molecular basis of rifampin resistance in M. tuberculosis and to use this information to design a DNA diagnostic assay for the rapid and specific detection of Rif strains of M. tuberculosis directly from clinical specimens. It is anticipated that use of this assay will make the identification of these drug-resistant organisms possible much earlier in the course of disease. To reach the goals of this research the following objectives will be accomplished: The mutation(s) which are associated with the development of rifampin resistance in M. tuberculosis will be determined using PCR amplification and a direct DNA sequencing methodology. The stability of these mutations will be examined using characterized clinical isolates of Rib M. tuberculosis. These isolates have been characterized using drug- susceptibility testing and restriction fragment length polymorphism analysis to differentiate strains. A DNA diagnostic assay for the detection of Rif M. tuberculosis strains using characterized clinical isolates will be developed. This assay is based on PCR amplification of a target sequence which contains all known Rif alleles and subsequent hybridization with allele-specific probes and nonisotopic detection. The feasibility of detecting Rif M. tuberculosis strains directly from clinical specimens using the DNA diagnostic assay will be determined initially using sediments from highly-characterized sputum samples. Finally, the molecular basis of rifampin resistance in M. tuberculosis as a function of an altered beta-subunit of the DNA-dependent RNA polymerase will be characterized by purifying RNA polymerase holoenzymes from Rif and Rif strains of M. tuberculosis, purifying the subunits of these enzymes and reconstituting Rif beta-subunit into Rif holoenzyme. The hybrid holoenzyme will be analyzed for the production of mRNA in the presence and absence of rifampin.